Epigenetic regulation of Thy-1 gene expression by histone modification is involved in lipopolysaccharide-induced lung fibroblast proliferation

Abstract

Lipopolysaccharide (LPS)-induced pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts. Epigenetic regulation of thymocyte differentiation antigen 1 (Thy-1) is associated with lung fibroblast phenotype transformation that results in aberrant cell proliferation. However, it is not clear whether the epigenetic regulation of Thy-1 expression is required for LPS-induced lung fibroblast proliferation. To address this issue and better understand the relative underlying mechanisms, we used mouse lung fibroblasts as model to observe the changes of Thy-1 expression and histone deacetylation after LPS challenge. The results showed that cellular DNA synthesis, measured by BrdU incorporation, was impacted less in the early stage (24 hrs) after the challenge of LPS, but significantly increased at 48 or 72 hrs after the challenge of LPS. Meanwhile, Thy-1 expression, which was detected by real-time PCR and Western blot, in lung fibroblasts decreased with increased time after LPS challenge and diminished at 72 hrs. We also found that the acetylation of either histone H3 or H4 decreased in the LPS-challenged lung fibroblasts. ChIP assay revealed that the acetylation of histone H4 (Ace-H4) decreased in the Thy-1 promoter region in response to LPS. In addition, all the above changes could be attenuated by depletion of TLR4 gene. Our studies indicate that epigenetic regulation of Thy-1 gene expression by histone modification is involved in LPS-induced lung fibroblast proliferation.

Fig. 1

Effect of lipopolysaccharide (LPS) on lung fibroblast proliferation. DNA synthesis in lung fibroblasts at 0, 6, 24, 48 and 72 hrs after LPS challenge was detected by BrdU assay. *P < 0.05 indicates significant difference of the absorbance (OD₄₅₀) between the LPS-challenged cells and the control cells at the same time-point. Points represent mean values (n = 3) and error bars represent SD.

Fig. 2

Dynamic changes of Thy-1 expression in lung fibroblast in response to lipopolysaccharide (LPS) challenge. The mRNA of Thy-1 was detected by real-time PCR (A) and protein expression of Thy-1 was examined by Western blot (B) in lung fibroblasts at 0, 6, 24, 48 and 72 hrs after LPS challenge (1 μg/ml). Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments. *P < 0.05 compared with the control group at the same time-point.

Fig. 3

Effect of TLR4 on TLR4 expression in lung fibroblast in response to lipopolysaccharide (LPS) challenge. The effect of siRNA-mediated TLR4 depletion (1 × 10⁸ TU/ml for 48 hrs) on the expression of TLR4 mRNA and protein in lung fibroblasts 72 hrs after LPS challenge by real-time PCR (A) and Western blot (B) respectively. Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments. *P < 0.05 compared with the negative control group (column 1). †P < 0.05 compared with the positive control group (column 2).

Fig. 4

Effect of TLR4 on Thy-1 expression in lung fibroblast in response to lipopolysaccharide (LPS) challenge. The effect of siRNA-mediated TLR4 depletion (1 × 10⁸ TU/ml for 48 hrs) on the expression of Thy-1 mRNA and protein in lung fibroblasts 72 hrs after LPS challenge by real-time PCR (A) and Western blot (B) respectively. Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments. *P < 0.05 compared with the negative control group (column 1). †P < 0.05 compared with the positive control group (column 2).

Fig. 5

Changes in histone modifications in lung fibroblast in response to lipopolysaccharide (LPS) challenge. Histone deacetylation in lung fibroblasts 72 hrs after LPS challenge was estimated according to the expression of Ace-H3 versus Total-H3 (A) and Ace-H4 versus Total-H4 (B) detected by Western blot. Histone modification presented by Ace-H4 (C) in the Thy-1 gene promoter region in lung fibroblasts 72 hrs after LPS challenge was detected by the quantitative ChIP assay. The effect of TLR4 on LPS-induced histone deacetylation was evaluated by siRNA-mediated TLR4 depletion (1 × 10⁸ TU/ml for 48 hrs). Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments. *P < 0.05 compared with the negative control group (column 1). †P < 0.05 compared with the positive control group (column 2).